Because alphabeta T cells recognize antigen as peptides associated with major histocompatibility complex (MHC) molecules on cell surfaces, understanding how protein antigens are transformed into peptides bound to MHC molecules and determining which cells present antigen in vivo are critical to understanding T cell immunity. MHC class II molecules assemble in the presence of invariant chain (Ii), which plays multiple roles in regulating the function of class II molecules. Recent experiments have shown that association of Ii with the chaperone calnexin prevents Ii degradation and retains it in this organelle for efficient association with MHC class II proteins. Other results support a new model in which Ii association with the class II binding site via CLIP plays a primary role in proper formation and transport of class II dimers, rather than acting to prevent endogenous antigen presentation. New data suggest that oligomerization of signals in the cytoplasmic tail of Ii is necessary for efficient targeting of these Ii-class II complexes to endosomes. Using transfected cells expressing various combinations of wild-type and mutant MHC class II and Ii molecules, we have found that Ii is essential for presentation of only some peptide determinants within a single protein antigen. Signals in the cytoplasmic tails of the MHC class II alpha and beta chains control Ii-independent presentation of other determinants. We have identified a dileucine-based motif as critical for this function, as well as determined that the absence of this signal affects antigen presentation by slowing endocytic entry of mature surface class II molecules. These data suggest that two separate pools of MHC class II molecules (newly synthesized and recycling, mature) provide for maximal capture of antigenic information. We have begun to develop new monoclonal antibody reagents capable of tracking and/or quantitating specific peptide-MHC class II complexes on isolated cells or in tissue sections. These investigations are providing a detailed picture of how and where MHC class II ligands able to stimulate CD4+ T cells are formed and made available for recognition. Intact extracellular proteins are usually not converted into peptides bound to MHC class I molecules, but some exceptions involving particulate antigen have been reported. Our studies suggest that this reflects the breach of the endosome membrane and entry of the antigen into the conventional cytoplasmic class I processing pathway. These data are of significance for vaccine design and may also bear on CD8+ T cells responses to some pathogens that normally reside in endocytic vesicles.